Cholinergic neurons are involved in numerous physiological functions in mammals including motor activity, sleep-wakefulness (vigilance) and cognitive behavior (cognition). The dramatic consequences of the loss of cholinergic neurons which innervate the hippocampus and the cerebral cortex, associated with Alzheimer's disease, are evidence of the importance of these neurons for normal behavior (Bierer et al., 1995). Impairment of the cholinergic system has been also reported in other pathologies, for example in certain forms of epilepsy and in schizophrenia (Holt et al., 1999; Friedman et al., 2007).
One way to investigate the function of cholinergic structures is to analyze the consequences of their impairment. Until recently, the generation of such a loss-of-function phenotype relied on the destruction of cholinergic neurons by methods which are not specific for these neurons (excitotoxins, electrolytic lesions).
The immunotoxin 192IgG-saporin is a more selective tool to deplete cholinergic neurons of the basal forebrain (Wiley et al., 1991; Waite et al., 1996; Wrenn et al., 1999), but when delivered by intracerebroventricular injection, it can also affect other cell populations expressing the p75 neurotrophin receptor, including the cerebellar Purkinje cells (Heckers et al., 1994; Walsh et al., 1995).
Another approach to generate cholinergic deficits is to inactivate specifically the cholinergic neurons in given nuclei. This strategy has the advantage of sparing the neurons and affecting only cholinergic neurotransmission. This can be achieved by inhibiting locally the expression of a protein required for cholinergic presynaptic function. Conditional gene knockout requires the slow and costly generation of mice with loxP sites and is restricted to date to this animal species. Constitutive knockout of the ChAT gene in the mouse is further lethal at birth and thus cannot be used to investigate cholinergic function in the adult (Brandon et al., 2003; Misgeld et al., 2002).
Inventors herein provide new and powerful tools that are able to achieve effective and specific inhibition of choline acetyltransferase expression which can be spatially and/or timely controlled. The method according to the present invention now obviates the generation of knock-out mice and further allows studies in species other than the mouse.
The present invention allows for the first time inactivation of cholinergic neurotransmission, without destroying cholinergic structures, using a viral vector, in particular a lentiviral vector which can efficiently transduce cells of the nervous system and specifically modulate (e.g., down-regulate or inhibit) the synthesis or expression of a presynaptic cholinergic protein, in particular of the choline acetyltransferase (ChAT), in these cells, through RNA interference (RNAi). Other presynaptic cholinergic proteins required for cholinergic function include the high affinity choline transporter (CHT1) and the vesicular acetylcholine transporter (VAChT).
The products of the invention, in particular the nucleic acid sequences and viruses, in particular lentiviruses, are new cholinergic antagonists which are, in particular, able to inhibit specifically ChAT synthesis in cells which produce acetylcholine in vitro, ex vivo or in vivo. Examples of cells producing acetylcholine are cells of the nervous system, in particular cholinergic neurons of the Central Nervous System (CNS), or cells of the Peripheral Nervous System (PNS) such as neurons located in the intestine. Cells producing acetylcholine may further be non neuronal cells such as lymphocytes or more generally cells located in the blood or in the placenta.
Using such a nucleic acid or virus, it is now possible to study the particular role of cholinergic nuclei in the brain and, more generally, the functional anatomy of the cholinergic system. The present invention indeed allows the persistent or long-term, and preferably spatially restricted, suppression of the effects of a particular cholinergic protein, as defined previously, which inactivates the cholinergic neurotransmission. Using in particular modified lentiviral vectors allows timed and thus temporary and reversible ChAT suppression.
Thanks to the herein described virus vectors, it is also possible to develop new animal models (in particular rat and mouse animal models) of brain disorders or of nervous system diseases, without being compromised by serious unwanted side effects as seen in the past due to the lack of specificity of the methods of lesion which destroyed the whole structures containing the cholinergic neurons. Using the tools and methods herein described, it is possible to identify molecular networks regulated by cholinergic neurotransmission.
The screening of new drugs as well as the development of safe and efficient prophylactic or therapeutic strategies are also now possible using said tools and methods.